The present invention relates to methods for detecting agents that cause or potentiate DNA damage and to molecules and transformed cells that may be usefully employed in such methods.
DNA damage is induced by a variety of agents such as ultraviolet light, X rays, free radicals, methylating agents and other mutagenic compounds. These agents may cause damage to the DNA that comprises the genetic code of an organism and cause mutations in genes. In microorganisms such mutations may lead to the evolution of new undesirable strains of the microorganism. For instance, antibiotic or herbicide resistant bacteria may arise. In animals these mutations can lead to carcinogenesis or may damage the gametes to give rise to congenital defects in offspring.
These DNA damaging agents may chemically modify the nucleotides that comprise DNA and may also break the phosphodiester bonds that link the nucleotides or disrupt association between bases (T-A or C-G). To counter the effect of these DNA damaging agents cells have evolved a number of mechanisms. For instance the SOS response in E. coli is a well characterised cellular response induced by DNA damage in which a series of proteins are expressed, including DNA repair enzymes, which repair the damaged DNA.
There are numerous circumstances when it is important to identify what agents may cause or potentiate DNA damage. It is particularly important to detect agents that cause DNA damage when assessing whether it is safe to expose a person to these agents. For instance a method of detecting these agents may be used as a mutagenesis assay for screening compounds that are candidate food additives, medicaments or cosmetics to assess whether or not the compound of interest induces DNA damage. Alternatively methods of detecting DNA damaging agents may be used to monitor for contamination of water supplies with pollutants that contain mutagenic compounds.
Various methods, such as the Ames Test, for determining the toxicity of an agent are known. More recent developments are disclosed in WO 95/00834 which relates to the use of a light emitting organism (particularly the bacterium Photobacterium phosphoreum) for measuring the toxicity of industrial effluents. WO 95/07463 discloses a gene construct formed from DNA encoding for Green Fluorescent Protein and DNA encoding for a regulatory element (such as a promoter induced by heavy metals) which may be used to detect pollution. However these developments do not disclose means of specifically monitoring for the presence of agents that may cause or potentiate DNA damage. Furthermore these known methods are not sensitive enough to detect agents that cause or potentiate DNA damage at low concentrations.
According to a first aspect of the present invention, there is provided a recombinant DNA molecule comprising a regulatory element that activates gene expression in response to DNA damage operatively linked to a DNA sequence that encodes a light emitting reporter protein.
According to a second aspect of the invention, there is provided a recombinant vector comprising a DNA molecule in accordance with the first aspect of the present invention and a DNA vector.
According to a third aspect of the invention, there is provided a cell containing a DNA molecule in accordance with the first aspect of the present invention or a recombinant vector in accordance with the second aspect of the present invention.
According to a fourth aspect of the present invention, there is provided a method of detecting for the presence of an agent that causes or potentiates DNA damage comprising subjecting a cell in accordance with the third aspect of the present invention to an agent and monitoring the expression of the light emitting reporter protein from the cell.
By xe2x80x9cregulatory elementxe2x80x9d we mean a DNA sequence which regulates the transcription of a gene with which it is associated.
By xe2x80x9coperatively linkedxe2x80x9d we mean that the regulatory element is able to induce the expression of the reporter protein.
By xe2x80x9creporter proteinxe2x80x9d we mean a protein which when expressed in response to the regulatory element of the DNA molecule of the invention is detectable by means of a suitable assay procedure.
The method of the fourth aspect of the invention is suitable for assessing whether or not an agent may cause DNA damage. It is particularly useful for detecting agents that cause DNA damage when assessing whether it is safe to expose a person to DNA damaging agents. For instance, the method may be used as a mutagenesis assay for screening whether or not known agents, such as candidate foodstuffs, medicarnents or cosmetics, induce DNA damage. Alternatively the method of the invention may be used to monitor for contamination of water supplies with pollutants containing DNA damaging agents.
The method of the fourth aspect of the invention may equally be used for assessing whether an agent may potentiate DNA damage. For example, certain agents can cause DNA damage by inhibiting DNA repair (for instance by preventing expression of a repair protein) without directly inflicting DNA damage. These agents are often known as co-mutagens and include agents such as lead.
The regulatory element of the DNA molecule of the first aspect of the invention activates expression of the reporter protein when DNA damage occurs. Such regulatory elements ideally comprise a promoter sequence which induces RNA polymerase to bind to the DNA molecule and start transcribing the DNA encoding for the reporter protein. The regulatory element may also comprise other functional DNA sequences such as translation initiation sequences for ribosome binding or DNA sequences that bind transcription factors which promote gene expression following DNA damage. Regulatory elements may even code for proteins which act to dislodge inhibitors of transcription from the regulated gene and thereby increase transcription of that gene.
Preferred regulatory elements are DNA sequences that are associated in nature with the regulation of the expression of DNA repair proteins. For instance, the regulatory elements from genes such as RAD2, RAD6, RAD7, RAD18, RAD23, RAD51, RAD54, CDC7, CDC8, CDC9, MAG1, PHR1, DIN1, DDR48, RNR1, RNR2, RNR3 and UB14 from yeast may be used to make molecules according to the first aspect of the invention. There are also regulatory elements associated with inducible excision repair genes in Neurospora, inducible recombinational repair genes in Ustilago and UV inducible irradiation damage recovery pathway genes in mammalian cells which may be used.
A preferred regulatory element comprises the promoter and 5xe2x80x2 regulatory sequences of the RAD54 repair gene. Such a regulatory element may be derived from yeast and particularly Saccharomyces cerevisiae. It is most preferred that the regulatory element comprises the promoter and 5xe2x80x2 regulatory sequences of the RAD54 repair gene which correspond to the DNA sequence identified as SEQ ID NO 1 or a functional analogue or fragment thereof.
Another preferred regulatory element comprises the promoter and 5xe2x80x2 regulatory sequences of the RNR2 gene. The RNR2 gene may be found on chromosome X of Saccharomyces cerevisiae. A preferred regulatory element may be derived from between co-ordinates 387100 and 398299 associated with the RNR2 gene on chromosome X as identified in the Saccharomyces cerevisiae genome database. The database may be accessed by the World Wide Web at many sites. For example at genome-www.stanford.edu.
The DNA sequences that encode a light emitting reporter protein may code for any light emitting protein, however it is preferred that the DNA sequences code for a protein that is fluorescent.
Preferred DNA sequences that encode a light emitting reporter protein code for Green Fluorescent Protein (GFP) and light emitting derivatives thereof. GFP is from the jelly fish Aquorea Victoria and is able to absorb blue light and re-emits an easily detectable green light and is thus suitable as a reporter protein. GFP may be advantageously used as a reporter protein because its measurement is simple and reagent free and the protein is non-toxic.
Derivatives of GFP include DNA sequences encoding for polypeptide analogues or polypeptide fragments of GFP which are able to emit light. Many of these derivatives absorb and re-emit light at wavelengths different to GFP found endogenously in Aquorea victoria. For instance, preferred DNA molecules according to the first aspect of the invention have a DNA sequence that encodes the S65T derivative of GFP (in which serine 65 of GFP is replaced by a threonine). S65T GFP has the advantage that it is brighter than wild-type GFP (when excited at its longest-wavelength peak) and shows only slow photobleaching. Furthermore S65T GFP produces a good quantum yield of fluorescence and matches the output of argon ion lasers used in fluorescence activated cell sorters. Cells according to the third aspect of the invention which contain DNA molecules coding S65T GFP may be used according to the method of the fourth aspect of the invention and are particularly useful when light emission is measured from cell extracts (see below).
Another preferred DNA sequence encodes for a yeast enhanced GFP (YEGFP) such as the GFP derivative described by Cormack et al. (1997) (in Microbiology 143 p303-311). Such YEGFP has an amino acid sequence which is biased for usage in yeast. Thus YEGFP is particularly suitable for transforming cells according to the third aspect of the invention which are yeast. Furthermore we have found that light emitted from YEGFP in such yeast is even greater than that emitted by S65T derivatives. For example, light output from yeast strain FF18984 (also known as Y486) transformed with a DNA molecule coding YEGFP was double that output from FF18984 transformed with a DNA molecule coding S65T GFP. We have found that YEGFP is particularly suited for use in methods according to the fourth aspect of the invention which involve monitoring light emission from intact cells according to the third aspect of the invention (discussed in more detail below).
DNA molecules coding YEGFP are also useful because YEGFP is less heat sensitive than nascent GFP.
Most preferred DNA molecules according to the first aspect of the invention comprise a RAD54 regulatory element operatively linked to a DNA sequence that encodes a GFP or light emitting derivative thereof.
The DNA molecule may be contained within a suitable DNA vector to form a recombinant vector according to the second aspect of the present invention. The vector may for example be a plasmid, cosmid or phage. Such recombinant vectors are of great utility when replicating the DNA molecule of the first aspect of the invention. Furthermore recombinant vectors are highly useful for transforming cells with the DNA molecule and may also promote expression of the reporter protein.
The recombinant vectors will frequently include one or more selectable markers to enable selection of cells transfected with the DNA vector and, preferably, to enable selection of cells harbouring the recombinant vectors that incorporate the DNA molecule of the first aspect of the invention. Examples of such selectable markers include genes conferring resistance to kanamycin (or G148) and ampicillin. Selectable markers may include those which restore prototrophy, for example the yeast URA3 gene.
Recombinant vectors may be designed such that the vector will autonomously replicate in the cytosol of the cell. In this case, elements which induce DNA replication may be required in the recombinant vector. A suitable element is derived from the 2xcexc plasmid. Such replicating vectors can give rise to multiple copies of the DNA molecule in a transformant and are therefore useful when over-expression (and thereby increased light emission) of the reporter protein is required.
Alternatively the recombinant vector may be designed such that the vector and DNA molecule of the first aspect of the invention integrate into the genome of a cell. Such integration has the advantage of improved stability compared to replicative plasmids In this case DNA sequences which favour targeted integration (e.g. by homologous recombination) are desirable. For example, incorporation into the recombinant vector of fragments of the HO gene from chromosome IV of Saccharomyces cerevisiae favours targeted integration in Saccharomyces cerevisiae or cell-lines derived therefrom. It is preferred that the fragment of the HO gene has the sequence identified as SEQ ID No 5 or is a derivative thereof. It is also possible to insert multiple copies of integrating recombinant vectors into the genome. This will allow enable greater expression and increase the signal output further. For instance, the vectors may be targeted to chromosome XII using sequences from the ribosomal DNA array.
Preferably recombinant vectors may be formed from PFA vectors or derivatives thereof which are known to the art (see Wach et al. (1994) Yeast 10 p1793-1808).
Preferred DNA molecules according to the first aspect of the invention have a DNA sequence that encodes for a GFP or light emitting derivative thereof that is derived from these PFA vectors.
Preferred recombinant vectors are PFA KANMX3GFP-RAD54, pWDH443 and pWDH444 which are described in detail in the Example. The preferred DNA molecules of the first aspect of the present invention which comprise a RAD54 regulatory element operatively linked to a DNA sequence that codes for a GFP or light emitting derivative thereof may be derived from these most preferred recombinant vectors PFA KANMX3GFP-RAD54, pWDH443 or pWDH444.
Recombinant vector PFA KANMX3GFP-RAD54 comprises the vector of sequence listing SEQ ID NO 4 with the regulatory element of sequence listing SEQ ID NO 1 or a functional analogue or fragment thereof inserted between the Pac1 and BamH1 restriction enzyme sites of the vector of sequence listing SEQ ID NO 4.
Preferred recombinant vector pWDH443 comprises PFA KANMX3GFP-RAD54 with a fragment of the HO gene corresponding to SEQ ID NO 5 inserted at the unique BamH1 site of the KANMX3GFP-RAD54. pWDH443 comprises DNA coding for S65T GFP and is capable of integrating into the yeast genome.
Preferred recombinant vector pWDH444 comprises a fragment of the 2xcexc plasmid ligated with the large fragment generated from the BamH1 and Pme1 digestion of pWDH443. The fragment of the 2xcexc plasmid may correspond precisely to the large HindIII/BamH1 fragment released from the plasmid pRDK249, described in the Journal Biological Chemistry, Volume 266, p14049, FIG. 1 (1991) in the article by Johnson, A. W. and Kolodner, R. D. The DNA from the 2xcexc plasmid in pWDH444 allows this recombinant vector to autonomously replicate in the cytosol of a host cell and thereby allows the recombinant vector to be present in high copy numbers (which can be advantageous when over-expression of GFP and increased light emitting capability is desired).
Other preferred recombinant vectors are yEGFP-443 and yEGFP-444 (see FIGS. 11 and 12).
yEGFP-443 is derived from pWDH443 and comprises the large fragment of pWDH443 generated by Pac1 and Asc1 digestion with the Pac1 and Asc1 digestion product of SEQ ID NO 6 (which encodes a YEGFP) ligated into the said large fragment yEGFP-443 comprises DNA coding for YEGFP and is capable of integrating into the yeast genome.
yEGFP-444 is derived from pWDH444 and comprises the large fragment of pWDH444 generated by Pac1 and Asc1 digestion with the Pac1 and Asc1 digestion product of SEQ ID NO 6 (which encodes a YEGFP) ligated into the said large fragment. The DNA from the 2xcexc plasmid in yEGFP-444 allows this recombinant vector to autonomously replicate in the cytosol of a host cell and thereby allows the recombinant vector to be present in high copy numbers (which can be advantageous when over-expression of GFP and increased light emitting capability is desired).
According to the third aspect of the invention the DNA molecule is incorporated within a cell. Such host cells may be prokaryotic or eukaryotic. Suitable host cells include bacteria, plant, yeasts, insect and mammalian cells. Preferred host cells are yeast cells such as Saccharomyces cerevisiae. Yeast are preferred because they can be easily manipulated like bacteria but are eukaryotic and therefore have DNA repair systems that are more closely related to humans than those of bacteria. Thus the use of such yeast in the method of the invention represents an improved method for detecting DNA damage relative to the Ames test. The Ames test uses bacteria (strains of Salmonella typhimurium) which when exposed to a putative DNA damaging agent may result in a genotoxicity result (positive or negative) which, because of the differences between prokaryotes and eukaryotes, would not necessarily be representative of the effects of such agents in eukaryotes such as humans.
Another benefit of using yeast cells as a host is that DNA repair systems are inducable in yeast unlike in humans where the repair systems are largely constitutive.
Preferred yeast cells include:
(i) Y485 in haploid form;
(ii) Y486 (also known as FF18984) in haploid form
(iii) Y485/486 in diploid form;
(iv) FY73
(v) YLR030wxcex1; and
(vi) Y300.
These strains may all be found in national yeast strain collections.
The type of yeast strain used can influence the DNA damage response and we have found that light emission can vary greatly depending upon the yeast strain used. In this respect we have found that (i), (ii) and (iii) above are particularly useful strains for use according to the method of the invention.
Host cells used for expression of the protein encoded by the DNA molecule of the invention are ideally stably transformed, although the use of unstably transformed (transient) cells is not precluded.
Transformed cells according to the third aspect of the invention may be formed according to following procedures. PFA vectors may be used as suitable starting material from which DNA molecules of the first aspect of the invention and recombinant vectors of the second aspect of the invention may be formed. The known PFA vectors contain, or may be manipulated to contain, DNA encoding for GFP and derivatives thereof. Such vectors may be manipulated, by known molecular biology techniques, to insert a suitable regulatory element adjacent to the GFP coding sequence thus forming a DNA molecule of the first aspect of the invention which is contained within a recombinant vector according to the second aspect of the invention. For instance, pWDH443, pWDH444, yEGFP-443 and yEGFP-444 may be derived from such PFA vectors. The DNA molecule may be excised from the recombinant vector, or more preferably the DNA molecule contained within the recombinant vector may be used to transform a cell and thereby form a cell according to the third aspect of the invention. The cell is ideally a yeast cell (for instance one of the abovedescribed strains). Such transformed cells may be used according to the method of the fourth aspect of the invention to assess whether or not agents induce or potentiate DNA damage. GFP expression is induced in response to DNA damage and the light emitted by GFP may be easily measured using a fluorimeter as an index of the DNA damage caused. For instance, the light emitted by GFP at 511 nm (after excitation between 475 and 495 nmxe2x80x94e.g. 488 nm) in response to DNA damage, may be evaluated either in a suspension of a defined number of whole cells or from a defined amount of material released from cells following breakage.
Many known methods of detecting DNA damage (including the Ames Test and related methods) assay lasting DNA damage, as an endpoint, either in the form of misrepaired DNA (mutations and recombinations) or unrepaired damage in the form of fragmented DNA. However most DNA damage is repaired before such an endpoint can be measured and lasting DNA damage only occurs if the conditions are so severe that the repair mechanisms have been saturated. Preferred methods of the fourth aspect of the present invention are much more sensitive than these known methods because they detect repair activity (which we have found to be detectable when actual DNA damage is undetectable) which prevents the above mentioned endpoint being reached. Therefore the method of the fourth aspect of the invention may be used to detect for the presence of DNA damaging agents or DNA damage potentiating agents at concentrations below the threshold for which actual DNA damage may be detected.
The method of the fourth aspect of the invention is particularly useful for detecting agents that induce DNA damage at low concentrations. The methods may be used to screen compounds, such as candidate medicaments, food additives or cosmetics, to assess whether it is safe to expose a living organism, particularly people, to such compounds.
Alternatively the methods of the fourth aspect of the invention may be employed to detect whether or not water supplies are contaminated by DNA damaging agents or agents that potentiate DNA damage. For instance, the methods may be used to monitor industrial effluents for the presence of pollutants that may lead to increased DNA damage in people or other organisms exposed to the pollution.
When the methods are used to detect whether or not water supplies are contaminated, the cells according to the third aspect of the invention are ideally unicellular organisms such as bacteria, algae, protoza and particularly yeast.
The expression of light emitting reporter protein may be monitored according to the method of the invention from cell extracts (in which case cells transformed with any of the abovedescribed recombinant DNA molecules and/or recombinant vectors may be used) or from samples containing intact, whole cells (in which case yEGFP-443 and yEGFP-444 transformed yeast cells are preferably used).
There are several advantages associated with the use of whole cells. As there is no requirement to break open cells, the number of treatment steps is reduced. The production of extracts requires cell-harvesting, washing, breakage with glass beads and centrifugation to clear the extract. The reduction in treatment steps also reduces the risk of errors arising in handling and makes the method much faster. Furthermore cell density and light emission can be made simultaneously giving greater sensitivity. Therefore the method of the invention is preferably performed by growing cells transformed with a recombinant vector according to the second aspect of the invention (such as yEGFP-4443 or yEGFP-444), incubating the cells with the agent which putatively causes DNA damage for a predetermined time and monitoring the expression of the light emitting reporter protein directly from a sample of the cells.
When whole cells are used they are preferably contained in low fluorescence growth medium. This can obviate the need to wash the cells before measurements are made and therefore reduce the number of steps in the method further. For instance, preferred yeast according to the third aspect of the invention may be grown in F1 medium (described in Walmsley et al. (1983) Mol. Gen. Genet. 192 p361-365).
According to a preferred embodiment of the method of the invention FF18984 cells may be transformed with yEGFP-444 and grown in F1 medium. A putative DNA damaging agent (e.g. a food additive or potential medicament or an agent contained within a water sample or effluent sample) may then be added to the F1 medium containing the cells. The cells are then allowed to grow for a defined period of time after which a sample of the cells is removed and fluorescence measured therefrom. This measurement may be effected by estimating the cell concentration and fluorescence in the sample using nephelometry (light scattering). For example, cells can be illuminated at 600 nm and the scattered light (at 600 nm) estimated at 90 degrees to the incident beam. The light emitted by GFP can be measured by excitation at 495 nm and fluorescent light emitted at 518 nm measured at 90 degrees to the incident beam. Both measurements may be made in a single cuvette. A normalised GFP light emission is calculated by dividing the GFP fluorescence value by the whole cell light scattering value (at 600 nm). This embodiment of the invention has the advantage that it may be easily carried out with a minimum of steps (i.e an incubation period followed by direct fluorescence measurement).
The method of the invention should ideally employ sensitive fluorimeters and reduce light scattering in order that light emission can be accurately measured from the reporter protein. We have found that sensitivity can be improved by using a 495 nm filter which is introduced between the sample chamber and the emission-detector of the fluorimeter. Such a filter further reduces the impact of light scattering and improves the sensitivity of the method when samples containing whole cells are used.